Keywords - regulation of membrane curvature and dynamics, regulation of dynamin-dependent endocytosis of E-cadherin during oogenesis, Cip4 acts downstream of Cdc42 to activate the WASP-WAVE-Arp2/3 pathway in the notum and the wing epithelium

Schultheis, D., Schwirz, J. and Frasch, M. (2019). RNAi screen in Tribolium reveals involvement of F-BAR proteins in myoblast fusion and visceral muscle morphogenesis in insects. G3 (Bethesda). PubMed ID: 30733382Summary:
In a large-scale RNAi screen in Tribolium castaneum for genes with knock-down phenotypes in the larval somatic musculature, one recurring phenotype was the appearance of larval muscle fibers that were significantly thinner than those in control animals. Several of the genes producing this knock-down phenotype corresponded to orthologs of Drosophila genes that are known to participate in myoblast fusion, particularly via their effects on actin polymerization. A new gene previously not implicated in myoblast fusion but displaying a similar thin-muscle knock-down phenotype was the Tribolium ortholog of Nostrin, which encodes an F-BAR and SH3 domain protein. Genetic studies of Nostrin and Cip4, a gene encoding a structurally related protein, in Drosophila show that the encoded F-BAR proteins jointly contribute to efficient myoblast fusion during larval muscle development. Together with the F-Bar protein Syndapin they are also required for normal embryonic midgut morphogenesis. In addition, Cip4 is required together with Nostrin during the profound remodeling of the midgut visceral musculature during metamorphosis. It is proposed that these F-Bar proteins help govern proper morphogenesis particularly of the longitudinal midgut muscles during metamorphosis.

Members of the Fes-CIP4 homology Bin-amphiphysin-Rvs161/167 (F-BAR) protein family form crescent-shaped dimers that are able to shape membranes into vesicles and tubules. F-BAR proteins have been grouped into six subfamilies, the Cdc42-interacting protein 4 (Cip4) subfamily, the Fes subfamily of non-receptor tyrosine kinases, the protein kinase C and casein kinase substrate in neurons protein (pacsin) subfamily, the Slit-Robo RhoGTPase-activating proteins (SrGAPs), the FCH-domain-only (FCHO) and the proline-serine-threonine phosphatase-interacting protein (PSTPIP) subfamilies (Ahmed, 2010; Heath, 2008). The phylogenetic subgrouping is mainly based on structural similarities of the N-terminal F-BAR module, and on the composition and architecture of C-terminal domains. Distinct differences of the intrinsic F-BAR domain curvature observed among the different F-BAR-domain proteins are thought to reflect characteristic preferences in sensing and/or inducing membrane invaginations of different curved geometry. Consistent with this idea, members of FCHO subfamily bind to very low membrane curvatures and are found to be essential for the initial step of membrane invagination in endocytosis (Henne, 2010). Other F-BAR proteins, such as members of the Cip4 subfamily, which includes the Cdc42-interacting protein 4 (Cip4), the transducer of Cdc42-dependent actin assembly (Toca-1) and the formin-binding protein 17 (FBP17), have a preference for higher membrane curvatures present in later steps during vesicle formation (Frost, 2008; Frost, 2009; Shimada, 2007; Zobel, 2015 and references therein).

Unlike those of the FCHO subfamily, Cip4 subfamily proteins contain a C-terminal SH3 domain that binds dynamin and factors that promote actin filament formation (Itoh, 2006; Tsujita, 2006). All three members of the Cip4 subfamily are able to activate N-WASP by promoting Arp2/3-mediated actin nucleation in vitro (Ho, 2004). Cip4 and Toca-1 also associate with Cdc42 through a central coiled-coil region (Aspenström, 2009). The current view is that Cip4-related proteins may stabilize plasma membrane invaginations and, subsequently, recruit dynamin and WASP proteins to the neck of endocytic pits that mediate the constriction and scission of vesicles. Recruitment of WASP proteins to newly formed vesicles also promotes the formation of actin comet tails that provide the driving force for endocytic vesicle movement (Bu, 2010; Fricke, 2009). However, understanding of how F-BAR proteins function in vivo in a physiological context is still incomplete because loss-of-function studies in higher organisms are limited. Mice that lack Cip4 are viable and show only a weak endocytosis defect of the insulin-responsive glucose transporter Glut4 (Feng, 2010). Mutant animals also display a reduced platelet production and defective integrin-dependent T-cell adhesion. Both defects are probably caused by decreased WASP-dependent actin polymerization, rather than impaired endocytosis (Chen, 2013). Given the mild phenotypes, the two other Cip4-like subfamily members Toca-1 and FBP17 might have redundant functions and could compensate for the loss of Cip4 function (Zobel, 2015).

Initial RNA interference (RNAi) studies in Drosophila melanogaster revealed that Cip4 regulates dynamin-dependent endocytosis of E-cadherin at adherens junctions (Leibfried, 2008). As in mammals, function of cip4 is not essential for fly development (Fricke, 2009). cip4 mutants show duplicated wing hairs because of an impaired endocytosis. Further analyses revealed that Cip4 acts downstream of Cdc42 to activate the WASP-WAVE-Arp2/3 pathway in the notum and the wing epithelium (Leibfried, 2008; Fricke, 2009). In addition, a postsynaptic, endocytosis-independent function of Cip4 has been identified at the neuromuscular junction. This function also depends on an interaction with the Cdc42-WASP-Arp2/3 pathway but does not require a functional F-BAR domain (Nahm, 2010; Zobel, 2015 and references therein).

The Drosophila genome contains an additional, not yet characterized gene encoding a Cip4-like F-BAR protein with highest similarities to human Nostrin (CG42388). Human Nostrin was originally identified as an interaction partner of the endothelial nitric oxide synthase (eNos, Zimmermann, 2002). Cell culture studies further suggest that Nostrin regulates N-WASP and/or dynamin-dependent trafficking and the activity of endothelial nitric oxide synthase (eNos) (Icking, 2005; Zimmermann, 2002). However, an in vivo role of Nostrin in the regulation of eNos activity or endocytosis has not yet been found. A recent loss-of-function study in zebrafish and mice revealed a role of Nostrin in endothelial cell morphology during vascular development (Kovacevic, 2012). Antisense morpholino oligonucleotide (MO)-mediated knockdown of Nostrin in developing zebrafish affects the migration of endothelial tip cells of intersegmental blood vessels (Kovacevic, 2012). Remarkably, nostrin-knockout mice are viable and show only mild retinal angiogenesis defects (Kovacevic, 2012). This suggests that other F-BAR proteins compensate for nostrin function in mutant mice (Zobel, 2015 and references therein).

As Drosophila contains only a single gene copy of each F-BAR subfamily (Fricke, 2010), studies in flies are well-suited to address putative functional redundancies within and between F-BAR domain subfamilies. This study presents a functional analysis of CG42388, which encodes the Drosophila Nostrin protein, and its physiological relationship to Cip4. Flies that lack Nostrin are viable and fertile. However, loss of both nostrin and cip4 results in reduced viability and fertility. Double mutant flies show a strong multiple wing hair phenotype and females are semi-sterile. Egg chambers of double mutant flies show strong encapsulation defects that are likely to be caused by an impaired membrane turnover of E-cadherin. Cip4 and Nostrin, preferentially, bind similar membrane curvatures but localize at distinct subdomains of membrane structures in cells. These data suggest an important, non-redundant function of Cip4 and Nostrin in the regulation of membrane dynamics in epithelial morphogenesis (Zobel, 2015).

F-BAR proteins play an important role in the regulation of membrane curvatures in a sequential manner during endocytosis (Suetsugu, 2010). Despite their pivotal functions in linking actin and membrane dynamics, recent single-knockout studies revealed that many F-BAR proteins are not essential for development and, thus, might have redundant or cooperative functions in vivo (Feng, 2010; Fricke, 2009). In fission yeast, the Cip4-like F-BAR proteins Cdc15 and Imp2 are examples for such synergistic function of two F-BAR proteins. Cells deficient for either Cdc15 or Imp2 show mild defects in cytokinesis but are still able to divide (Demeter, 1998; Fankhauser, 1995). However, deletion of both C-terminal SH3 domains of the proteins completely restricts the division of the cells (Roberts-Galbraith, 2009; Zobel, 2015 and references therein).

This study provides first evidence for cooperative function of the two F-BAR proteins Cip4 and Nostrin in the multicellular context of Drosophila development. Like Cip4, members of the Nostrin subfamily show a remarkably high evolutionary conservation and single orthologs can be found from porifera to humans. All the more surprising is the fact that nostrin loss-of-function mutant flies have no obvious phenotype. Only after removal of both cip4 and nostrin, was a strong enhancement found of the phenotypic traits already observed in cip4 single mutants. Double mutants show a substantial reduction in the number of offspring. Both mutant females and males display reduced fertility, indicating a common function of both F-BAR proteins in early morphogenesis. Female sterility of double mutant flies is caused by strong defects in egg chamber morphogenesis. The formation of compound egg chambers results from a defective encapsulation in the germarium, a phenotype that has not yet been described for many mutants. Most mutations that have been reported of so far, either affect gene functions directly through the regulation of cell division or control of follicle cell differentiation through Notch/Delta signaling. However, multicyst egg chambers in nost;;cip4 double mutants display neither defects in cell division nor in the differentiation of follicle cells (Zobel, 2015).

Interestingly, loss of Maelstrom (Mael), a high-mobility group box protein that regulates microtubule organization leads to egg chambers with cell division defects but also results in an encapsulation defect with misplaced oocytes that is similar to the one observed in nost;;cip4 double mutants. Mael forms a complex with the components of the microtubule-organizing center (MTOC) -- including centrosomin and γ-tubulin, which seems to be required not only for early oocyte determination but also egg chamber packing and oocyte positioning in the germarium. Interestingly, in mael mutant multicyst egg chambers E-cadherin is not enriched on the oocyte cortex and not apically concentrated in the follicle epithelium as in wild type. nost;;cip4 double mutant egg chambers show a similar E-cadherin mislocalization, suggesting that the microtuble cytoskeleton plays an important role in E-cadherin localization. Consistently, Nostrin mainly localizes to Rab11-positive vesicles that move along microtubules. Thus, Nostrin might act on Rab11-dependent E-cadherin trafficking along microtubules. A strong requirement for Rab11 in E-cadherin trafficking in germline stem cells (GSC) and in the maintenance of GSC identity has recently been identified. Mosaic egg chambers are severely disorganized, comprising mispositioned oocytes. Most importantly, compound egg chambers can be found that contain two or more germline cysts surrounded by a single continuous follicle epithelium, as was observed in this study for nost;;cip4 mutants. However, rab11-null follicle stem cells (FSC) give rise to the normal number of cells that enter polar, stalk and epithelial cell differentiation pathways. Like Rab11, Nostrin and Cip4 functions do not seem to be required in follicle cell differentiation (Zobel, 2015).

Given the dramatic switch in Nostrin expression in the germline cysts between region 2a and region 2b, when protein levels drop from highest to very low or no expression, an important function is proposed of Nostrin in germ cells rather than in somatic follicle cells. The germline cyst undergoes a dramatic change in shape within this region, as is reflected by a transformation from a spherical to a lens-shaped structure. This morphological transition might imply important changes in the adhesiveness mediated by homophilic E-cadherin cell-cell contacts between germ cells within the cyst. Thus, it is proposed that Nostrin and Cip4 are involved in the regulation of this transition by controlling E-cadherin endocytosis and vesicle recycling in germline cells. A failure of nost;;cip4 mutant cysts in adopting a lens-shaped morphology might interfere with their encapsulation by follicle cells, which results in the formation of compound egg chambers (Zobel, 2015).

Given the substantial mislocalization of E-cadherin that becomes obvious in double mutant egg chambers, an additional role of both F-BAR proteins in the maintenance of E-cadherin cell-cell contacts between germline and somatic follicle cells is suggested (Zobel, 2015).

Cip4 and Nostrin partially mark the same membrane structures but they localize to distinct subregions in vivo. Consistently, in vitro liposome studies showed that both proteins prefer defined membrane curvatures of similar diameter, i.e. both might associate with similarly shaped membrane compartments. However, the different appearance of Cip4- and Nostrin-decorated vesiculo-tubular structures might also reflect differences in lattice formation of these two F-BAR domain proteins. It is hypothesized that these regular arrays of electron-dense structures at liposomes represent regular Cip4 lattices formed upon self-association by head-to-tail and lateral interactions as previously supposed by real-space reconstruction (Frost, 2008). Because such patterns were not observed at tubular structures following incubation in the presence of Nostrin, it is suggested that Nostrin does not polymerize into rigid helical coats that are thought to be the structural basis for membrane invagination. Consistently, unlike Cip4, overexpression of Nostrin in S2R+ cells did not induce membrane tubulation (Fricke, 2009). How do Cip4 and Nostrin cooperate in membrane remodeling and vesicle trafficking? In cells, Cip4 mainly localizes to Rab5-positive early endosomes (Fricke, 2009), whereas Nostrin marks both Rab5- and Rab11-positive vesicles. Thus, a cooperative recruitment model is proposed, in which first Cip4 promotes membrane invagination, vesicle scission and motility of Rab5-positive membrane compartments by recruiting dynamin and the WASP-WAVE-Arp2/3 pathway. Nostrin will then be recruited to Cip4-positive membrane structures because Nostrin prefers the curvature induced by the highly organized Cip4 coats. Yet, at these membrane compartments, both proteins still occur in a spatially segregated manner, as visualized in EM analyses of Cip4- and Nostrin-coated membrane tubules. This segregation might reflect that Nostrin is not interacting with Cip4, and that Cip4 has the ability to bind PE - which Nostrin does not have. Furthermore Nostrin protein arrays seem less organized, as reflected by the broader range of curvatures induced in vitro and by the lack of regular structures of Nostrin-coated membranes. Strong formation of rigid lattices might explain why Cip4 tubulates membranes effectively in vitro and in vivo, and why anti-Cip4 labeling usually outlines tubular structures. In contrast, Nostrin is confined to Cip4-free segments of these structures and predominantly appears at the end of such Cip4-coated tubules because the end does not accommodate a Cip4 coat optimized for cylindrical surfaces (Zobel, 2015).

Interestingly, Kif13A (a kinesin motor that directly binds Rab11) is most enriched at the tips of membrane tubules (Delevoye, 2014). Moreover, Kif13A localizes to distinct Rab11-positive subdomains within sorting endosomes and is thought to initiate the formation of recycling endosomal tubules along microtubules through its motor activity (Delevoye, 2014). Interestingly, Nostrin directly interacts with the Drosophila Kif13A homologue Khc73 and colocalizes with Khc73-marked endosomes that move along microtubules. A close link between Nostrins and kinesin motors seem to be evolutionarily conserved and the interaction is likely mediated by a conserved bipartite tryptophan-based kinesin-1 binding motif (Dodding, 2011). Thus, in the current model of cooperative recruitment, Cip4 stabilizes endosomal tubules, whereas Nostrin defines subdomains of recycling intermediates of endosomal tubules and makes contact with microtubules through the kinesin Khc-73 for long-range trafficking of recycling endosomes (Zobel, 2015).

A similar scenario might also take place during cell polarization of the wing epithelium. Here, Cip4 and Nostrin act together to control the polarized outgrowth of a single actin-rich protrusion called prehair, a process that also requires tight coupling of membrane trafficking and the cytoskeleton. The restriction of wing hair formation at the most distal apical vertex of each wing cell depends on the Frizzled-PCP signaling pathway. A key step in the cell polarization is the asymmetric localization of core PCP proteins at adjacent cell membranes within the plane of the epithelium. Thus, one of the central questions in understanding PCP signaling is how this asymmetric localization is achieved. Based on live-imaging studies, selective endocytosis and directional transport of Frizzled along polarized non-centrosomal microtubules have been proposed as possible mechanisms for asymmetric polarization. Previous studies that had used microtubule antagonists already revealed an important role of the microtubule cytoskeleton in order to localize prehair initiation to the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. Multiple pre-hair formation is also caused by overexpression of Frizzled, presumably through an ectopic activation of the pre-hair nucleation machinery. However, the multiple wing hair phenotype in nost;;cip4 double mutant wings does not seem to affect the asymmetric distribution of Frizzled. Interestingly, a similar Frizzled-independent multiple wing hair phenotype has recently been observed in mutants that affect casein kinase 1γ (CK1γ, also known as CSNK1G). Loss of CK1γ disrupts the apical localization of Rab11 at the base of prehairs, suggesting that Ck1γ regulates Rab11-mediated polarized vesicle trafficking that is required for prehair nucleation. Consistently, expression of either a dominant-negative or a dominant-active Rab11 variant strongly induces the formation of multiple wing hairs. Overexpression of Cip4 or Nostrin alone also phenocopies the multiple wing hair defect of nost;;cip4 double mutants. Like CK1γ and Rab11, Cip4 and Nostrin accumulate at the base of forming prehairs. Because Nostrin colocalizes with Rab11-positive vesicles that move along microtubules, it is proposed that Nostrin is involved in Rab11-mediated polarized vesicle trafficking in the developing wing (Zobel, 2015).

Polarized Rab11-dependent vesicle trafficking of E-cadherin is also needed for the hexagonal packing of wing cells. During this process, irregularly shaped cells adopt a hexagonal geometry by coordinated endocytosis and Rab11-dependent recycling of junctional E-cadherin. Hexagonal packing starts shortly after pupal molt and ends just before wing hair formation but, remarkably, also depends on components of the PCP pathway. The underlying molecular mechanism that links hexagonal packing and hair formation in the wing is unknown. However, both processes depend on vesicle trafficking because suppression of Rab11, Rab23 or the simultaneous knockdown of cip4 and nostrin results not only in multiple wing hairs but also affects the regular hexagonal array of wing epithelial cells. A similar E-cadherin-dependent process of cell packing and remodeling can also be observed in the dorsal thorax, an epithelium that originally derived from the fused proximal parts of two wing imaginal discs. A role in E-cadherin membrane turnover has already been reported for Cip4 in the developing thorax epithelium of Drosophila (Leibfried, 2008). In cells that express cip4 dsRNA, E-cadherin-GFP accumulates in apical punctate structures and elongated malformed tubules form at the cell cortex (Leibfried, 2008). These long and defective endocytic structures do not tolerate fixation and could only be observed in live-imaging experiments. This study observed an even stronger defect on E-cadherin membrane dynamics upon simultaneous downregulation of Cip4 and Nostrin. The number of elongated malformed tubules that form at the cell cortex is clearly increased. Moreover, knockdown of both cip4 and nostrin cause obvious defects in the formation of E-cadherin junctions, a phenotype that was never observed when suppressing either cip4 or nostrin. These strong junctional defects might be responsible for the lethality of late pupae following RNAi transgene expression by the aptereous-Gal4 driver. Thus, it is concluded that both F-BAR proteins play an important cooperative rather than a redundant function in E-cadherin trafficking and junction maintenance (Zobel, 2015).

Developmental processes are intimately tied to signaling events that integrate the dynamic reorganization of the actin cytoskeleton and membrane dynamics. The F-BAR-domain-containing proteins are prime candidates to couple actin dynamics and membrane trafficking in different morphogenetic processes. This study presents the functional analysis of the Drosophila F-BAR protein Cip4/Toca1 (Cdc42-interacting protein 4/transducer of Cdc42-dependent actin assembly 1). Cip4 is able to form a complex with WASP and SCAR/WAVE and recruits both actin-nucleation-promoting factors to invaginating membranes and endocytic vesicles. Actin-comet-tail-based movement of these vesicles depends not only on WASP but largely on WAVE function. In vivo, loss of cip4 function causes multiple wing hairs. A similar phenotype is observed when vesicle scission is affected after Dynamin suppression. Gene dosage experiments show that Cip4 and WAVE functionally interact to restrict wing hair formation. Further rescue experiments confirm that Cip4 is able to act through WAVE and WASP in vivo. Biochemical and functional data support a model in which Cdc42 acts upstream of Cip4 and recruits not only WASP but also SCAR/WAVE via Abi to control Dynamin-dependent cell polarization in the wing. It is concluded that Cip4 integrates membrane trafficking and actin dynamics through WASP and WAVE. First, Cip4 promotes membrane invaginations and triggers the vesicle scission by recruiting Dynamin to the neck of nascent vesicles. Second, Cip4 recruits WASP and WAVE proteins to induce actin polymerization, supporting vesicle scission and providing the force for vesicle movement (Fricke, 2009).

Despite the detailed structural and biochemical knowledge about the mode of action of F-BAR proteins, understanding how these proteins might function in the cellular and developmental context is still quite limited. This study analyzed the role of the Drosophila F-BAR protein Cip4/Toca-1 in S2R+ cell culture as well as in the developmental context of the fly. This study provides the first evidence that Cip4/Toca-1 is able to integrate membrane trafficking and actin dynamics not only through WASP but in particular through WAVE. The results support a model in which Cdc42 acts upstream of Cip4 and recruits WAVE via Abi to shape wing hair morphogenesis (Fricke, 2009).

It is proposed that Cip4 acts at two different steps of endocytosis during wing epithelium polarization. First, Cip4 promotes membrane invaginations and triggers the scission by recruiting Dynamin to the neck of nascent vesicles. A block of this essential early endocytic step by suppression of Dynamin or Cip4 function results in similar phenotypes. This observation highlights the importance of endocytic trafficking for wing hair formation. Subsequently, Cip4 recruits WAVE to induce actin polymerization, providing the force for vesicle movement. A block of both processes, loss of membrane invagination mediated by Cip4 and decreased membrane motility due to the loss of the membrane-associated F-actin results in stronger defects and therefore a stronger-wing-hair phenotype. Following this assumption, enhanced actin polymerization after overexpression of WAVE or WASP can indeed compensate for the lack of Cip4 function in promoting membrane invagination. These data indicate that WASP and WAVE are largely interchangeable, although endogenous WASP and WAVE proteins regulate Arp2/3-mediated actin polymerization in a cell-specific manner (Fricke, 2009).

In conclusion, Cip4 is in a central position to control the formation of F-actin via recruitment and subsequent activation of WAVE. Moreover, because a tissue-specific interaction of Cip4 with WASP regulating the endocytosis of E-cadherin was recently demonstrated, it is concluded that Cip4 and Cdc42 form distinct functional complexes with WASP and WAVE in a tissue-specific manner to couple F-actin formation to membrane remodeling (Fricke, 2009).

Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4

) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).

Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).

Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).

The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).

In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).